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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
immunology and microbiology
A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue
Journal of Virological Methods, Volume 149, No. 1, Year 2008
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Description
A high-throughput method of isolating and cloning geminivirus genomes from dried plant material, by combining an Extract-n-Amp™-based DNA isolation technique with rolling circle amplification (RCA) of viral DNA, is presented. Using this method an attempt was made to isolate and clone full geminivirus genomes/genome components from 102 plant samples, including dried leaves stored at room temperature for between 6 months and 10 years, with an average hands-on-time to RCA-ready DNA of 15 min per 20 samples. While storage of dried leaves for up to 6 months did not appreciably decrease cloning success rates relative to those achieved with fresh samples, efficiency of the method decreased with increasing storage time. However, it was still possible to clone virus genomes from 47% of 10-year-old samples. To illustrate the utility of this simple method for high-throughput geminivirus diversity studies, six Maize streak virus genomes, an Abutilon mosaic virus DNA-B component and the DNA-A component of a previously unidentified New Word begomovirus species were fully sequenced. Genomic clones of the 69 other viruses were verified as such by end sequencing. This method should be extremely useful for the study of any circular DNA plant viruses with genome component lengths smaller than the maximum size amplifiable by RCA. © 2008 Elsevier B.V. All rights reserved.
Authors & Co-Authors
Shepherd, Dionne Natalie
South Africa, Cape Town
University of Cape Town
Martin, Darren Patrick
South Africa, Cape Town
University of Cape Town
Lefeuvre, Pierre F.
France, Saint-denis
Peuplements Végétaux et Bioagresseurs en Milieu Tropical Pvbmt
Monjane, Adérito Luis
South Africa, Cape Town
University of Cape Town
Owor, Betty Elizabeth
South Africa, Cape Town
University of Cape Town
Rybicki, Edward P.
South Africa, Cape Town
University of Cape Town
Varsani, Arvind
South Africa, Cape Town
University of Cape Town
Statistics
Citations: 130
Authors: 7
Affiliations: 2
Identifiers
Doi:
10.1016/j.jviromet.2007.12.014
ISSN:
01660934
Research Areas
Genetics And Genomics